T-cell epitope peptides

ABSTRACT

The T-cell epitope site on a Japanese cypress (hinoki) pollen allergen molecule has been identified by stimulating a T-cell line established from a patient suffering from Japanese cypress pollen allergy with an overlap peptide covering the primary structure of the Japanese cypress pollen allergen. The peptide is useful in peptide-based immunotherapy for patients with spring tree pollinosis including patients with Japanese cypress pollinosis having cross reactivity with Japanese cypress pollen. The peptide is also useful for diagnosing spring tree pollinosis.

This application is a divisional, and claims priority, of co-pendingU.S. application Ser. No. 09/202,464, filed Mar. 9, 1999, which claimspriority of International Application No. PCT/JP97/02031, filed Jun. 12,1997. The disclosures of U.S. application Ser. No. 09/202,464 andInternational Application No. PCT/JP97/02031 are incorporated herein byreference in their entirety.

TECHNICAL FIELD

The present invention relates to T-cell epitope peptides of pollenallergen and a composition for peptide-based immunotherapy comprisingthe peptides as effective ingredients. This composition is useful fortreating and/or preventing pollinosis in springtime.

BACKGROUND ART

About 10% of the Japanese population suffers from pollinosis developedin springtime such as cedar pollinosis. This condition has been on theincrease and is attracting public attention.

The period when pollinosis is developed generally corresponds to theperiod when pollens scatter. In many cases, symptoms of pollinosis stillremain after the season in which cedar pollens scatter because mostpatients with cedar pollinosis are also sensitized with Japanese cypresspollens (Hiroki cypress pollens) that start to scatter just after thecedar pollen-scattering period. Thus, patients who are also sensitive toJapanese cypress pollens suffer from the symptoms of pollinosis for asignificant portion of the year.

Cedar pollens and Japanese cypress pollens possess common antigenicity(Takeshi Ide et al., Allergy Clinic 11, 174-178, 1991). Thecross-reactivity of IgE antibodies between cedar pollens and Japanesecypress pollens has been established (Taniai M. et al., Mol. Immunol.30, 183-189, 1993). The positivity index of patients with springpollinosis for their allergen-specific IgE antibodies is 83.5% for cedarpollens, 80.0% for Japanese cypress pollens, and 76.4% for both pollens(Mitsuhiro Okano et al., Allergy 43, 1179-1184, 1994). In addition, 60%of the patients with cedar pollinosis possess Japanese cypresspollen-specific IgE antibodies (Yozo Saito, Chiryo (Therapy) 78,1571-1576, 1996). Based on these reports, it is generally recognizedthat cedar pollinosis patients can develop pollinosis to Japanesecypress pollens and vice versa.

Pollinosis is a typical immediate type I allergy induced by anantigen-antibody reaction between a pollen allergen (which is an antigencausing allergy and is substantially the same as an antigen) and an IgEantibody specific to the allergen. Thus, pollinosis is now prevented andtreated using methods theoretically based on the mechanism by which typeI allergies develop. This mechanism is briefly described below.

An antigen that has invaded the body is presented to helper T cells byantigen-presenting cells. As a result, B cells mature intoantibody-producing cells. The antibody-producing cells produce anantigen-specific IgE antibody, which binds to the surface of mast cells.A subsequently invading antigen binds to the IgE antibody on the mastcells. This stimulation releases chemical mediators like histamine fromthe mast cells, thereby causing an allergic symptom.

The following three methods are mainly used to prevent and treatallergies based on the above mechanism: 1) evasion of an antigen thatcauses allergy, 2) chemotherapy typically using an anti-histaminic, and3) desensitization therapy using an allergen. However, method 1) isdifficult to implement practically, and method 2) is merely symptomatictherapy. Method 3) is expected to be the only treatment attacking theroot problem, but it is not always effective and may cause serious sideeffects such as anaphylactic shock.

For these reasons, peptide-based immunotherapy using T-cell epitopepeptides of allergen has been recently attempted to prevent and treatallergies. T-cell epitopes participate in initiating and retaining animmune response to a protein allergen that causes clinical symptoms ofallergies. These T-cell epitopes bind to HLA class II molecules on thesurface of antigen-presenting cells to stimulate the related T-cellsubpopulation. The stimulation is thought to trigger an initial responseat the helper T-cell level. This initial response causes proliferationof T cells, secretion of lymphokines, a localized inflammatory response,migration of proliferated immune cells to the inflammatory sites, andactivation of the B-cell cascade that precedes antibody production. IgEantibodies that are isotypes of these antibodies are critical to thedevelopment and retention of allergies. Furthermore, their production isinfluenced by the properties of lymphokines secreted by helper T cellsat the beginning of the above-described cascade. The T-cell epitope is abasic element or the minimum unit to be recognized by a T-cell receptor.This epitope contains amino acid sequence necessary to recognize thereceptor. Allergic inflammation can be treated by controlling theresponse of the helper T cell, which plays a key role inimmunosuppression, using the T-cell epitope peptide.

Known therapeutic agents for allergies using T-cell epitope peptidesinclude a therapeutic composition comprising a T-cell epitope peptide ofcat-origin allergen (a PCT application published in Japan (JP-WA) No.Hei 7-505365), a therapeutic composition comprising a T-cell epitopepeptide of cedar pollen Cry j 1 (JP-WA-Hei 8-502163), and amulti-epitope peptide obtained by joining T-cell epitopes of cedarpollens Cry j 1 and Cry j 2 (Japanese Patent Application No. Hei8-80702). The main allergen of Japanese cypress pollen, Cha o 1, isreported to have molecular weights of 45 KD or 50 KD. Each molecule hasthe same isoelectric point of 6.8 and consists of a protein containing5% carbohydrate (Takeshi Ide, et al., Nippon Kafun Gakkaishi (Journal ofthe Japanese Pollen Association) 34, 39, 1988). However, their primarystructures were unknown, and accordingly, no T-cell epitope site hasbeen identified on the allergen molecules yet. Recently, the presentinventors succeeded in cloning the Japanese cypress pollen allergengene, and clarified that, in addition to Cha o 1, another type of theallergen, Cha o 2, was present. Furthermore, the primary structures ofCha o 1 and Cha o 2 were determined (Japanese Patent Application No. Hei6-335089).

DISCLOSURE OF THE INVENTION

The period when cedar pollen scatter overlaps that of Japanese cypresspollen is referred to as the mixed pollen-scattering period. These twopollens possess a common antigenicity, which makes it difficult todistinguish symptoms caused by cedar pollens from those caused byJapanese cypress pollens. The symptoms sometimes continue or developeven after the cedar pollen-scattering period. Since pollens found inthe air during that period are mostly Japanese cypress pollens, thesesymptoms seem to be caused by Japanese cypress pollens. Since moreJapanese cypress trees are planted than cedar trees, the amount ofscattered Japanese cypress pollen is increasing year after year and willexceed that of cedar pollens in the near future. It is thus desirable toestablish a method for preventing and treating allergies based on theroot overall pollinosis caused by tree pollens in springtime, includingJapanese cypress pollinosis and cedar pollinosis. Peptide-basedimmunotherapy using T-cell epitope peptides is expected to lead toallergy treatment based on the root pollinosis. As described above,several methods for such immunotherapy are known for cedar pollinosis.However, nothing has been reported on Japanese cypress pollinosis or onpollinosis caused by tree pollens in springtime, including cedar andJapanese cypress pollens.

An objective of the present invention is to provide T-cell epitopepeptides useful for peptide-based immunotherapy for Japanese cypresspollinosis. Another objective of the present invention is to provideT-cell epitope peptides useful for peptide-based immunotherapy forpatients with pollinosis caused by tree pollens in springtime includingpatients with cedar pollinosis who show a cross-reactivity with Japanesecypress pollens.

The present inventors have identified a T-cell epitope site on theallergen molecules of Japanese cypress pollen by stimulating a T-cellline established from patients with Japanese cypress pollinosis withsynthetic overlapping peptides that cover the entire primary structureof Japanese cypress pollen allergens, thus solving the above problems.

The present invention is comprised of the inventions described in eachclaim and will be described below in more detail.

The present inventors determined the amino acid sequence (described inJapanese Patent Application No. Hei 6-335089) of the major allergen, Chao 1 (mature protein), of Japanese cypress pollen allergen shown as SEQID NO: 1 and that of Cha o 2 shown as SEQ ID NO: 2. The amino acidsequence of Cha o 1 has 80% homology to cedar pollen allergen Cry j 1,and that of Cha o 2 has 75% homology to cedar pollen allergen Cry j 2.

A number of amino acid substitutions are observed in the allergensderived from pollens, mites, and bee venom. These allergen species arecalled isoallergens. For example, eleven isoallergens have been isolatedfrom birch tree pollen Bet v I, and their amino acid sequences differfrom each other within a range of 2 to 15% (Swoboda, I. et al., J. Biol.Chem. 270: 2607-2613, 1995). At present, two isoallergens, in which sixamino acid residues are substituted in a mature protein region, havebeen found in Cry j 2 (unexamined published Japanese Patent Applications(JP-A) No. Hei 8-47392 and No. Hei 7-170986). One skilled in the art canreasonably expect that isoallergens would be present in Cha o 1 and Chao 2 as well. Such isoallergens are also included in Cha o 1 and Cha o 2referred to in the present invention.

The family of cedar trees is classified into nine genera, and the familyof Japanese cypress, into seven genera. It is reported that allergensfrom Cryptomeria, Redwood, and Metasequoia, which belong to the cedar(Taxodiaceae) family, and Umbrella Pine, which is hypothesized to belongto either an independent family, the cedar family, or the pine family,show cross-reactivity with those from Japanese Cypress, Sawara Cypress,Oriental Arbor-vitae, Needle Juniper and Chinese Juniper, which belongto the family of Cupressaceae (Takeshi Ide, et al., Allergy Clinic, 11,174-178, 1991). In view of this report, cedar allergens are broadlycross-reactive with the allergens of Japanese cypress. Therefore, thepeptides of the present invention are generally effective not only forJapanese cypress pollinosis but also for cedar pollinosis as well.

To obtain the T-cell epitope peptides of the present invention,overlapping peptides that cover the entire primary structures of Cha o 1and Cha o 2 were synthesized; each peptide consists of the adequatenumber of amino acid residues (12 to 20 residues). The peptide of thepresent invention stimulates and/or suppresses the activity of T cellsderived from patients with pollinosis caused by tree pollens inspringtime. In other words, the peptide of the present invention caninduce proliferation of T cells or responses of T cells such assecretion of lymphokines, and/or can induce T-cell anergy(non-responsiveness). T-cell epitope sites on the allergen molecules canbe identified using T-cell growth as an index in accordance with themethod described in JP-A-Hei 8-47392. In particular, T-cell lines orT-cell clones, which are specifically reactive with Cha o 1 and Cha o 2,are established for every patient from peripheral lymphocytes of apatient with Japanese cypress pollinosis. The T-cell lines or T-cellclones are cultured in the presence of each peptide of the overlappingpeptides. The epitope sites are identified by measuring theproliferation of T cells in the presence of the peptide (e.g., uptake of[³H]thymidine into the cells) and calculating a stimulation index. Thestimulation index (SI) used herein is obtained by dividing theradioactive level of [³H]thymidine (cpm) taken up into the cells in thepresence of the peptide by the level of [³H]thymidine (cpm) taken upinto the cells in the absence of the peptide (control). Based on thedata obtained thus, a mean stimulation index for each peptide iscalculated for each patient group. The peptides found to induce T-cellresponse and/or induce T-cell anergy are defined as having T-cellstimulating activity. The preferable T-cell epitope peptides of thepresent invention possess a T-cell stimulating activity and thus containat least one T-cell epitope. Examples of the T-cell epitope peptide ofCha o 1 shown in FIG. 1 (specifically shown in FIG. 2, FIG. 3, and SEQID NO: 3 through SEQ ID NO: 37) include Peptide #1-2 (SEQ ID NO: 4),Peptide #1-4 (SEQ ID NO: 6), Peptide #1-5 (SEQ ID NO: 7), Peptide #1-6(SEQ ID NO: 8), Peptide #1-7 (SEQ ID NO: 9), Peptide #1-8 (SEQ ID NO:10) Peptide #1-10 (SEQ ID NO: 12), Peptide #1-11 (SEQ ID NO: 13),Peptide #1-12 (SEQ ID NO: 14), Peptide #1-14 (SEQ ID NO: 16), Peptide#1-15 (SEQ ID NO: 17), Peptide #1-16 (SEQ ID NO: 18), Peptide #1-19 (SEQID NO: 21), Peptide #1-20 (SEQ ID NO: 22), Peptide #1-21 (SEQ ID NO:23), Peptide #1-22 (SEQ ID NO: 24), Peptide #1-23 (SEQ ID NO: 25),Peptide #1-24 (SEQ ID NO: 26), Peptide #1-25 (SEQ ID NO: 27), Peptide#1-26 (SEQ ID NO: 28), Peptide #1-27 (SEQ ID NO: 29), Peptide #1-30 (SEQID NO: 32), Peptide #1-31 (SEQ ID NO: 33), Peptide #1-32 (SEQ ID NO:34), Peptide #1-33 (SEQ ID NO: 35), and Peptide #1-34 (SEQ ID NO: 36)(FIG. 4). Examples of the T-cell epitope peptide of Cha o 2 shown inFIG. 5 (specifically shown in FIG. 6, FIG. 7, and SEQ ID NO: 38 throughSEQ ID NO: 88) include Peptide #2-5 (SEQ ID NO: 42), Peptide #2-7 (SEQID NO: 44), Peptide #2-8 (SEQ ID NO: 45), Peptide #2-9 (SEQ ID NO: 46),Peptide #2-10 (SEQ ID NO: 47), Peptide #2-11 (SEQ ID NO: 48), Peptide#2-12 (SEQ ID NO: 49), Peptide #2-13 (SEQ ID NO: 50), Peptide #2-14 (SEQID NO: 51), Peptide #2-15 (SEQ ID NO: 52), Peptide #2-16 (SEQ ID NO:53), Peptide #2-17 (SEQ ID NO: 54), Peptide #2-18 (SEQ ID NO: 55),Peptide #2-19 (SEQ ID NO: 56), Peptide #2-20 (SEQ ID NO: 57), Peptide#2-21 (SEQ ID NO: 58), Peptide #2-22 (SEQ ID NO: 59), Peptide #2-23 (SEQID NO: 60), Peptide #2-24 (SEQ ID NO: 61), Peptide #2-25 (SEQ ID NO:62), Peptide #2-26 (SEQ ID NO: 63), Peptide #2-27 (SEQ ID NO: 64),Peptide #2-30 (SEQ ID NO: 67), Peptide #2-31 (SEQ ID NO: 68), Peptide#2-32 (SEQ ID NO: 69), Peptide #2-33 (SEQ ID NO: 70), Peptide #2-34 (SEQID NO: 71), Peptide #2-35 (SEQ ID NO: 72), Peptide #2-36 (SEQ ID NO:73), Peptide #2-37 (SEQ ID NO: 74), Peptide #2-38 (SEQ ID NO: 75),Peptide #2-40 (SEQ ID NO: 77), Peptide #2-41 (SEQ ID NO: 78), Peptide#2-42 (SEQ ID NO: 79), and Peptide #2-43 (SEQ ID NO: 80) (FIG. 8). Morepreferably, the T-cell epitope peptides have a mean stimulation index of2.0 or more. Examples include Peptide #1-2 (SEQ ID NO: 4), Peptide #1-7(SEQ ID NO: 9), Peptide #1-8 (SEQ ID NO: 10), Peptide #1-20 (SEQ ID NO:22), Peptide #1-22 (SEQ ID NO: 24), Peptide #1-24 (SEQ ID NO: 26),Peptide #1-26 (SEQ ID NO: 28), Peptide #1-32 (SEQ ID NO: 34), Peptide#1-33 (SEQ ID NO: 35), and Peptide #1-34 (SEQ ID NO: 36), which areshown in FIG. 1, and Peptide #2-10 (SEQ ID NO: 47), Peptide #2-20 (SEQID NO: 57), Peptide #2-21 (SEQ ID NO: 58), Peptide #2-40 (SEQ ID NO:77), Peptide #2-41 (SEQ ID NO: 78), Peptide #2-42 (SEQ ID NO: 79), andPeptide #2-43 (SEQ ID NO: 80), which are shown in FIG. 5. Mostpreferably, the T-cell epitope peptide has a minimum positivity index of100. Examples thereof include Peptide#1-7 (SEQ ID NO: 9), Peptide#1-22(SEQ ID NO: 24), Peptide #1-32 (SEQ ID NO: 34), and Peptide #1-33 (SEQID NO: 35), which are shown in FIG. 1, and Peptide #2-10 (SEQ ID NO:47), Peptide #2-20 (SEQ ID NO: 57), Peptide #2-40 (SEQ ID NO: 77),Peptide #2-41 (SEQ ID NO: 78), Peptide #2-42 (SEQ ID NO: 79), andPeptide #2-43 (SEQ ID NO: 80), which are shown in FIG. 5. The“positivity index” used herein is obtained by multiplying a meanstimulation index of a peptide by appearance frequency (%) of patientsshowing a T-cell response to the peptide.

To identify the epitope accurately, a peptide having the T-cellstimulating activity and thus containing at least one T-cell epitope maybe modified by deleting any of the amino acid residues at the aminoterminus or the carboxyl terminus of the peptide. The modified peptidemay then be examined for any change in the T-cell stimulating activity.When two or more peptides that share the overlapping region exhibit theT-cell stimulating activity, a new T-cell epitope peptide containing allor part of the overlapping peptides is prepared, and its T-cellstimulating activity is measured in the same manner.

The T-cell epitope peptide of the present invention may beimmunologically associated with Cry j 1 or Cry j 2 in the T-cellcross-reactivity. Specifically, 1) the amino acid sequence of Cha o 1has 80% homology to that of Cry j 1, and the amino acid sequence of Chao 2 has 75% homology to that of Cry j 2; 2) the amino acid sequence ofT-cell epitope peptide #1-2 of Cha o 1 (corresponding to amino acids11-30, SEQ ID NO:4, of mature type Cha o 1), which was identified inExample 5 of the present invention, is identical with the amino acidsequence of T-cell epitope peptide CJI-2 of Cry j 1 (corresponding toamino acids 11-30, of mature type Cry j 1; see FIG. 13 of JP-A-Hei8-502163) except for two amino acid residues (Ala at position 12 of Chao 1 corresponds to Ser of CJI-2, and Asp at position 15 of Cha o 1corresponds to Ala of CJI-2); and 3) both cedar pollens and Japanesecypress pollens have a common antigenicity. For these reasons, theorigin of the T-cell epitope of the present invention is not limited toJapanese cypress. The T-cell epitope peptide of the present invention iseffective not only for Japanese cypress pollinosis but also for cedarpollinosis.

In the T-cell epitope peptide of the present invention, the amino acidresidues that participate in recognizing the T-cell receptor can bedetermined by a known method (for example, measuring the change in theT-cell stimulating activity which might occur due to the substitution ofamino acid residues). The amino acid residues found to be essential foran interaction with the T-cell receptor are substituted with other aminoacid residues to antigen-specifically control the T-cell stimulatingactivity so that allergic inflammation can be suppressed (increase thereactivity of T cells, alter the lymphokine-producing pattern, anergyetc.). It has been reported that, when one amino acid residue at theT-cell recognition site of the T-cell epitope peptide of cedar pollenCry j 1 was substituted with another amino acid residue (substitutingThr at position 399 with Val) in a human allergy model, the resultinganalog peptide showed substantially the same T-cell growth and IL-4production as those of a wild type peptide, but showed increasedproduction of IFN-γ that suppressed the production of IgE antibodies(Ikagawa, S. et al., J. Aller. Clin. Immunol. 97, 54-64, 1996). It hasfurther been revealed that a binding motif of HLA class II moleculesconsists of three to five amino acid residues arranged via one or twointermediary amino acid residues. When these residues consist of severalkinds of specified amino acids, the peptide binds to the HLA class IImolecules (Matsushita, S. et al., J. Exp. Med. 180: 877-883, 1994).Therefore, allergic inflammation can be prevented by determining theamino acid residues of the T-cell epitope peptide of the presentinvention, which are essential for the interaction with HLA class IImolecules, by a known method, and substituting the relevant amino acidresidues with other amino acid residues. Furthermore, the T-cell epitopepeptide of the present invention can be modified so as to improve itssolubility, thereby increasing its therapeutic or preventing effects orstability. Such modification includes substitution, deletion, andaddition of the amino acid residues.

In the present invention, the T-cell epitope peptide preferably does notbind to IgE antibodies. Even if it binds to the IgE antibodies, thedegree of binding is substantially lower than that of binding of theallergen of natural Japanese cypress pollens, from which the peptide isderived, to the antibodies.

The T-cell epitope peptide of the present invention preferably containsat least seven amino acid residues. These regions may be joined via alinker such as Arg-Arg or Lys-Lys that is sensitive to cleavage with anenzyme such as cathepsin or trypsin to enhance the sensitivity toprocessing by antigen-presenting cells. Thus, a peptide region can beproduced to contain one or more T-cell epitopes. The T-cell epitopepeptide of the present invention may be used in combination with otherpeptides such as a T-cell epitope peptide of Cry j 1 (JP-WA-Hei8-502163) and/or a T-cell epitope peptide of Cry j 2 (JP-WA-Hei8-47392).

When a peptide containing at least one T-cell epitope peptide of thepresent invention is administered to an individual sensitive to Japanesecypress pollens and/or an individual sensitive to both Japanese cypressand cedar pollens, the peptide can control the individual's allergicresponse to the allergen(s) Such a peptide is thus effective forpeptide-based immunotherapy. In particular, the T-cell epitope peptideof the present invention in combination with the T-cell epitope peptideof cedar pollen is more effective for peptide-based immunotherapy for apatient with pollinosis caused by tree pollens in springtime,represented by cedar and Japanese cypress pollens.

The T-cell epitope peptide of the present invention may be used as adiagnostic tool for pollinosis caused by Japanese cypress pollenallergens or other tree pollens that are immunologically cross-reactivewith Japanese cypress pollen allergens. In such an application, theT-cell epitope peptide of the present invention is added to peripherallymphocytes collected from a patient in an amount of about 0.1 μg/ml toabout 1 mg/ml, and preferably about 1 to about 300 μg/ml. After themixture is incubated for a week, uptake of [³H]thymidine into thelymphocytes is assayed and assessed for diagnosis of pollinosis. TheT-cell epitope peptide of the present invention may also be used toevaluate either the function of T cells or proliferation of T cells orboth.

When the T-cell epitope peptide of the present invention is synthesizedusing recombinant DNA technology, host cells transformed with a nucleicacid containing a sequence coding for the peptide are cultured in amedium suitable for growing the host cells. The peptide can be harvestedfrom the culture supernatant or from the host cells by a method known inthe art. E. coli, yeasts, or mammal cells can be used as such hostcells.

When the T-cell epitope peptide of the present invention is used inpeptide-based immunotherapy for patients with pollinosis, the peptidemay be administered together with pharmaceutically acceptable diluentsor carriers. The “patient with pollinosis” as used herein includespatients with cedar pollinosis who show immunological cross-reactivitywith the allergen of Japanese cypress pollen. The T-cell epitope peptideof the present invention can be administered in a simple manner, forexample, by injection (subcutaneous or intravenous), instillation,rhinenchysis, oral administration, inhalation, or percutaneousadministration. In the case of injection, a single dose of the peptideranges preferably from about 1 μg to about 30 mg, and more preferablyfrom about 20 μg to about 10 mg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows T-cell epitope peptides of the Japanese cypress pollenallergen, Cha o 1, and a positivity index of each peptide.

FIG. 2 shows overlapping peptides (#1-1 (SEQ ID NO:3) to #1-28 (SEQ IDNO:30) of Cha o 1.

FIG. 3 shows overlapping peptides (#1-29 (SEQ ID NO:31) to #1-35 (SEQ IDNO:37)) of Cha o 1.

FIG. 4 shows peptides containing T-cell epitopes of Cha o 1.

FIG. 5 shows T-cell epitope peptides of Japanese cypress pollenallergen, Cha o 2, and a positivity index of each peptide.

FIG. 6 shows overlapping peptides (#2-1 (SEQ ID NO:38) to #2-27 (SEQ IDNO:64)) of Cha o 2.

FIG. 7 shows overlapping peptides (#2-28 (SEQ ID NO: 65) to #2-51 (SEQID NO:88)) of Cha o 2.

FIG. 8 shows peptides containing T-cell epitopes of Cha o 2.

BEST MODE FOR IMPLEMENTING THE INVENTION

Examples of the present invention will be described below, but are notto be construed to limit the scope of the present invention.

EXAMPLE 1 Synthesis of Overlapping Peptides

Based on the amino acid sequences of Japanese cypress pollen allergensCha o 1 (SEQ ID NO: 1) and Cha o 2 (SEQ ID NO: 2), overlapping peptidesconsisting of 20 amino acid residues (14 residues in Peptide #1-35 (SEQID NO: 37) and Peptide #2-51 (SEQ ID NO: 88), each containing 10overlapping residues) were synthesized by the Fmoc method using apeptide synthesizer (PSSM-8, Shimadzu Seisakusho Ltd.). Thirty-fiveoverlapping peptides were prepared for Cha o 1 (FIG. 1, SEQ ID NO: 3through SEQ ID NO: 37), and 51, for Cha o 2 (FIG. 5, SEQ ID NO: 38through SEQ ID NO: 88). The synthesized peptides were all purified byhigh-performance liquid chromatography (HPLC) using an ODS column. Thepurity was 90% or higher in all of the peptides. The molecular weightsof the purified peptides were identified by using a LASERMAT 2000(Finnigan MAT Ltd.).

EXAMPLE 2 Expression of the Recombinant Proteins in E. coli

Using a PCR technique, cDNA was amplified from plasmid DNA, in which Chao 1 cDNA or Cha o 2 cDNA encoding a Japanese cypress pollen antigen hadbeen cloned (Japanese Patent Application No. Hei 6-335089). Arestriction enzyme recognition site was attached to the terminus of eachcDNA. This DNA fragment was inserted into a histidine-tagged proteinexpression vector, pQE9, and the resulting vector was used to transformE. coli M15 (pREP4). Expression of the transforming gene was confirmedfor ampicillin-resistant clones by SDS-polyacrylamide gelelectrophoresis. The protein expressed was purified using a Ni-NTAagarose affinity column.

EXAMPLE 3 Establishment of T-Cell Line

A T-cell line on Cha o 1 was established as follows. Peripherallymphocytes collected from 19 patients found positive to Japanesecypress pollinosis using Ala STAT (Nippon DPC Corporation) or CAP-RAST(Pharmacia) were separated by specific gravity centrifugation usingFicoll-Paque. The lymphocytes (2×10⁶ cells) were suspended in RPMI 1640medium (GIBCO, Inc.) supplemented with 2 ml of plasma from the samepatient (10%) or human AB type serum (20%, Banpoh Tsusho Co., Ltd.). Thesuspension was incubated in a 24-well plate for 3 to 10 days (37° C.,CO₂ incubator, TABAI, Inc.), together with 10 to 30 μg/ml of therecombinant Cha o 1 obtained in Example 2 or with a mixture of theoverlapping peptides (0.01 to 1 μM) obtained in Example 1. When T cellsactivated by Cha o 1 stimulation were verified microscopically, 5 U/mlof IL-2 (Boehringer Mannheim) was added to the system, followed byincubation overnight. On the next day, the medium was replaced withfresh RPMI 1640 medium supplemented with 20 U/ml of IL-2, 10% or 20%human AB type serum. Incubation was continued for about 10 days with themedium being replaced every day in the same manner. The resulting T-cellline was examined for its specificity, and a sample of the T-cell linewas frozen and stored. A T-cell line stimulated by Cha o 2 was alsoestablished from 20 patients with Japanese cypress pollinosis in thesame way.

EXAMPLE 4 Establishment of Antigen-Presenting Cells

A lymphoblastoid cell line (B cell line) transformed by infecting Blymphocytes with EB virus (Epstein-Barr virus, EBV) was established toserve as antigen-presenting cells. First, EBV-producing B-95-8 cells(marmoset, ATCC CRL 1612) were cultured in RPMI 1640 medium supplementedwith 20% inactivated fetal calf serum (FCS, GIBCO Inc.). The culturesupernatant was filtered through a 0.22 μm sterile filter. The filtratewas frozen and stored at −80° C. Next, 1 ml of EBV solution was added tolymphocytes (2×10⁶ cells) of a patient with Japanese cypress pollinosis,and the mixture was maintained at 37° C. for 30 minutes for infection.The EBV-infected cells were washed twice and then incubated for about 20days in 20% FCS-RPMI 1640 medium supplemented with a final concentrationof 200 ng/ml of Cyclosporin (Sandoz Pharmaceutical Co., Ltd.). After thecell mass was observable by the naked eye, incubation was continued in20% FCS-RPMI 1640 medium for another 20 days. The resulting cells werefrozen and stored until they were used.

EXAMPLE 5 Identification of T-Cell Epitope Peptide

The cultured B cell line established in Example 4 was treated with 50μg/ml of mitomycin C (Sandoz Pharmaceutical Co., Ltd.) for 30 minutes orexposed to an X ray (50 g ray), followed by washing four times with RPMI1640 medium. After the B cells were inoculated on a 96-well plate(10,000 cells/well), the recombinant Cha o 1 or Cha o 2 was addedthereto in a final concentration of 10 g/ml. To the control group wasadded a hemolytic streptococcus cell wall antigen (SCW) in a finalconcentration of 10 μg/ml, Candida albicans antigen (CA) in a finalconcentration of 10 μg/ml, and a Tuberculin antigen (PPD) in a finalconcentration of 1 μg/ml). Subsequently, the T-cell line (20,000cells/well) from the same patient, whose B cell line had beenestablished, was inoculated into each well. After a 48-hour incubation,0.5 μCi [³H]thymidine was added to each well, and incubation wascontinued for a further 16 hours. After the cells were collected on aglass filter using a cell harvester (Berthold), uptake of [³H]thymidineinto the cells was measured with a liquid scintillation counter toconfirm the cell growth response.

After the T-cell line was confirmed to have proliferated specifically inresponse to Chao 1 or Cha o2, the growth response of the T-cell line toeach of the overlapping peptides (final concentration of 1 μM) wasexamined in the same manner as above using the T-cell line establishedin Example 3. A mean stimulation index of the T-cell line in growthresponse to the overlapping peptides, an appearance frequency, and apositivity index calculated therefrom are shown in FIGS. 1 and 5.

In addition, growth response of the T-cell line (N=17) to modifiedsequences (SEQ ID NO: 89and NO: 90) that corresponded to the amino acidsequences #2-11 and #2-12 in which one amino acid residue had beensubstituted, was examined. These two modified sequences exhibited T-cellstimulating activity of 1.6 and 1.2 in terms of the stimulation index,16% and 11% in terms of the appearance frequency, and 25.6 and 13.2 interms of the positivity index. As demonstrated above, the T-cell epitopepeptide of the present invention retained its T-cell stimulatingactivity even when one or more amino acid residues were mutated, and theactivity was enhanced in some cases.

INDUSTRIAL APPLICABILITY

The present invention provides peptides containing at least one T-cellepitope of Cha o 1 or Cha o 2, which are major allergens of Japanesecypress pollens. The present invention further includes a peptidefragment of other tree pollens showing immunological T-cellcross-reactivity with the peptides. These peptides are effective forpeptide-based immunotherapy of pollinosis caused by tree pollens inspringtime as represented by cedar and Japanese cypress pollens.

1.-10. (canceled)
 11. A peptide derived from Japanese cypress pollenallergen Cha o 2, wherein the peptide consists of: (i) (a) an amino acidsequence selected from the group consisting of SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ IDNO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ IDNO:64, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, said amino acidsequence having T-cell stimulating activity; or (b) a fragment of theamino acid sequence of (i) (a), the fragment being selected fromfragments of the amino acid sequences listed in (i) (a), wherein each ofthe fragments has T-cell stimulating activity equivalent to that of thecorresponding amino acid sequence of (i) (a); or (ii) a combination ofsequences, the sequences being selected from the amino acid sequenceslisted in (i) (a) and the amino acid sequence fragments recited in (i)(b).
 12. A composition comprising the peptide of claim 11, as an activeingredient, and a pharmaceutically acceptable diluent or carrier. 13.The composition of claim 12, wherein said composition can reduce thesymptoms of Japanese cypress pollinosis or cedar pollinosis in apatient.
 14. A peptide derived from Japanese cypress pollen allergen Chao 2, wherein the peptide consists of a combination of two or more aminoacid sequences and a linker sensitive to enzyme cleavage between eachamino acid sequence, wherein said two or more amino acid sequences ofthe combination are: sequences selected from the group consisting of SEQID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ IDNO:63, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ IDNO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, thesequences having T-cell stimulating activity; or fragments of thesequences, each fragment having T-cell stimulating activity equivalentto that of the corresponding sequence.
 15. The peptide of claim 14,wherein said linker is Arg-Arg or Lys-Lys.
 16. A composition comprisingthe peptide of claim 14, as an active ingredient, and a pharmaceuticallyacceptable diluent or carrier.
 17. The composition of claim 16, whereinsaid composition can reduce the symptoms of Japanese cypress pollinosisor cedar pollinosis in a patient.
 18. The peptide of claim 11, whereinthe peptide consists of: (A) an amino acid sequence selected fromsequences of the group consisting of: SEQ ID NO: 42, SEQ ID NO: 44, SEQID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49,SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO:54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ IDNO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQID NO: 64, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70,SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO:75, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80; or(B) a combination of amino acid sequences selected from the sequenceslisted in (A).
 19. The peptide of claim 11, wherein the peptide consistsof: (x) an amino acid sequence selected from sequences of the groupconsisting of SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO:77, SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80; or (y) acombination of amino acid sequences selected from the sequences listedin (x).
 20. The peptide of claim 11, wherein the peptide consists of:(X) an amino acid sequence selected from sequences of the groupconsisting of SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 77, SEQ ID NO:78, SEQ ID NO: 79, and SEQ ID NO: 80; or (Y) a combination of amino acidsequences selected from the sequences listed in (X).
 21. A compositioncomprising the peptide of claim 18 as an active ingredient, and apharmaceutically acceptable diluent or carrier.
 22. A compositioncomprising the peptide of claim 19 as an active ingredient, and apharmaceutically acceptable diluent or carrier.
 23. A compositioncomprising the peptide of claim 20 as an active ingredient, and apharmaceutically acceptable diluent or carrier.
 24. A method fortreating pollinosis caused by tree pollen in springtime, the methodcomprising administering the peptide of claim 11 to a patient that haspollinosis in the pollen-scattering season.
 25. A method for treatingpollinosis caused by tree pollen in springtime, the method comprisingadministering the peptide of claim 14 to a patient that has pollinosisin the pollen-scattering season.
 26. A method of diagnosing pollinosis,the method comprising: (a) providing a population of cells from anindividual, the population of cells comprising lymphocytes; (b)contacting said population of cells with a peptide of claim 11, and (c)detecting stimulation of the lymphocytes in response to the peptide asan indication that the individual is susceptible to pollinosis caused byJapanese cypress pollen allergens or by trees pollen allergens that areimmunologically cross-reactive with Japanese cypress pollen allergens.27. A method of diagnosing pollinosis, the method comprising: (a)providing a population of cells from an individual, the population ofcells comprising lymphocytes; (b) contacting said population of cellswith a peptide of claim 14, and (c) detecting stimulation of thelymphocytes in response to the peptide as an indication that theindividual is susceptible to pollinosis caused by Japanese cypresspollen allergens or by trees pollen allergens that are immunologicallycross-reactive with Japanese cypress pollen allergens.